A general problem in vaccinology has been an inability to generate high levels of CD8 T cells by immunization. This has impeded the development of vaccines against several diseases including malaria.
Plasmodium falciparum malaria causes hundreds of millions of malaria infections each year and is responsible for 1-2 million deaths annually. The development of an effective vaccine against malaria is thus a major priority for global public health. A considerable body of immunological research over the last twenty years had led to the identification both of candidate vaccine antigens from the parasite and immunological mechanisms on the host that are likely to protect against infection and disease. However, despite this progress there is still no means of vaccinating against malaria infection which has been shown to be effective in field trials.
A major problem has been the identification of a means of inducing a sufficiently strong immune response in vaccinated individuals to protect against infection and disease. So, although many malaria antigens are known that might be useful in vaccinating against malaria the problem has been how to deliver such antigens or fragments of them known as epitopes, which are recognized by cells of the immune system, in a way that induces a sufficiently strong immune response of a particular type.
It has been known for many years that it is possible to protect individuals by immunizing them with very large doses of irradiated malaria sporozoite given by bites from infected mosquitoes. Although this is a wholly impractical method of mass vaccination it has provided a model for analyzing the immune responses that might be mediating protective immunity against sporozoite infection (Nardin and Nussenzweig 1993).
A considerable amount of research over the last decade or more has indicated that a major protective immune response against the early pre-erythrocytic stage of P. falciparum malaria is mediated by T lymphocytes of the CD8+ ve type (CD8+ T cells). Such cells have been shown to mediate protection directly in mouse models of malaria infection (Nardin and Nussenzweig 1993). Such T cells have also been identified in individuals naturally exposed to malaria and in volunteers immunized with irradiated sporozoite (Hill et al. 1991; Aidoo et al. 1995; Wizel et al. 1995). There is much indirect evidence that such CD8+ T cells are protective against malaria infection and disease in humans (Lalvani et al. 1994).
CD8+ T cells may function in more than one way. The best known function is the killing or lysis of target cells bearing peptide antigen in the context of an MHC class I molecule. Hence these cells are often termed cytotoxic T lymphocytes (CTL). However, another function, perhaps of greater protective relevance in malaria infections is the ability of CD8+ T cells to secrete interferon gamma (IFN-xcex3). Thus assays of lytic activity and of IFN-xcex3 release are both of value in measuring a CD8+ T cell immune response. In malaria these CD8+ ve cells can protect by killing the parasite at the early intrahepatic stage of malaria infection before any symptoms of disease are produced (Seguin et al. 1994).
The agent of fatal human malaria, P. falciparum infects a restricted number of host species: humans, chimpanzees and some species of New World monkey. The best non-human model of malaria is the chimpanzee because this species is closely related to humans and liver-stage infection is observed consistently unlike in the monkey hosts (Thomas et al. 1994). Because of the expense and limited availability of chimpanzees most laboratory studies of malaria are performed in mice, using the rodent malaria species P. berghei or P. yoelii. These latter two models are well studied and it has been shown in both that CD8+ ve lymphocytes play a key role in protective immunity against sporozoite challenge.
Previous studies have assessed a large variety of means of inducing CD8+ T cell responses against malaria. Several of these have shown some level of CD8+ T cell response and partial protection against malaria infection in the rodent models (e.g. Li et al. 1993; Sedegah et al. 1994; Lanar et al. 1996). However, an effective means of immunizing with subunit vaccines by the induction of sufficiently high levels of CD8+ T lymphocytes to protect effectively against malaria sporozoite infection has not previously been demonstrated.
In recent years improved immune responses generated to potential vaccines have been sought by varying the vectors used to deliver the antigen. There is evidence that in some instances antibody responses are improved by using two different vectors administered sequentially as prime and boost. A variety of combinations of prime and boost have been tested in different potential vaccine regimes.
Leong et al. (Vaccines 1995, 327-331) describe immunizing mice firstly to DNA expressing the influenza haemagglutinin (HA) antigen and then with a recombinant fowlpox vector expressing HA. An enhanced antibody response was obtained following boosting.
Richmond et al. (Virology 1997, 230: 265-274) describe attempts to raise neutralizing antibodies against HIV-1 env using DNA priming and recombinant vaccinia virus boosting. Only low levels of antibody responses were observed with this prime boost regime and the results were considered disappointing.
Fuller et al. (Vaccine 1997, 15:924-926 and Immunol Cell Biol 1997, 75:389-396) describe an enhancement of antibody responses to DNA immunization of macaques by using a booster immunization with replicating recombinant vaccinia viruses. However, this did not translate into enhanced protective efficacy as a greater reduction in viral burden and attenuation of CD4 T cell loss was seen in the DNA primed and boosted animals.
Hodge et al. (Vaccine 1997, 15: 759-768) describe the induction of lymphoproliferative T cell responses in a mouse model for cancer using human carcinoembryonic antigen (CEA) expressed in a recombinant fowl pox virus (ALVAC). The authors primed an immune response with CEA-recombinant replication competent vaccinia viruses of the Wyeth or WR strain and boosted the response with CEA-recombinant ALVAC. This led to an increase in T cell proliferation but did not result in enhanced protective efficacy if compared to three wild type recombinant immunizations (100% protection), three recombinant ALVAC-CEA immunizations (70% protection) or WR prime followed by two ALVAC-CEA immunizations (63% protection).
Thus some studies of heterologous prime-boost combination have found some enhancement of antibody and lymphoproliferative responses but no significant effect on protective efficacy in an animal model. CD8 T cells were not measured in these studies. The limited enhancement of antibody response probably simply reflects the fact that antibodies to the priming immunogen will often reduce the immunogenicity of a second immunization with the same immunogen, while boosting with a different carrier will in part overcome this problem. This mechanism would not be expected to be significantly affected by the order of immunization.
Evidence that a heterologous prime boost immunization regime might affect CD8 T cell responses was provided by Li et al. (1993). They described partial protective efficacy induced in mice against malaria sporozoite challenge by administering two live viral vectors, a recombinant replicating influenza virus followed by a recombinant replicating vaccinia virus encoding a malaria epitope. Reversing the order of immunization led to loss of all protective efficacy and the authors suggested that this might be related to infection of liver cells by vaccinia, resulting in localization of CTLs in the liver to protect against the hepatocytic stages of malaria parasites.
Rodrigues et al. (J. Immunol. 1994, 4636-4648) describe immunizing mice with repeated doses of a recombinant influenza virus expressing an immunodominant B cell epitope of the malarial circumsporozoite (CS) protein followed by a recombinant vaccinia virus booster. The use of a wild type vaccinia strain and an attenuated but replication-competent vaccinia strain in the booster yielded very similar levels of partial protection. However the attenuated but replication competent strain was slightly less immunogenic for priming CD8 T cells than the wild type vaccinia strain.
Murata et al. (Cell. Immunol. 1996, 173: 96-107) reported enhanced CD8 T cell responses after priming with replicating recombinant influenza viruses and boosting with a replicating strain of vaccinia virus and suggested that the partial protection observed in the two earlier studies was attributable to this enhanced CD8 T cell induction.
Thus these three studies together provide evidence that a booster immunization with a replicating recombinant vaccinia virus may enhance to some degree CD8 T cell induction following priming with a replicating recombinant influenza virus. However, there are two limitations to these findings in terms of their potential usefulness. Firstly, the immunogenicity induced was only sufficient to achieve partial protection against malaria and even this was dependent on a highly immunogenic priming immunization with an unusual replicating recombinant influenza virus. Secondly, because of the potential and documented side-effects of using these replicating viruses as immunogens these recombinant vectors are not suitable for general human use as vaccines.
Modified vaccinia virus Ankara (MVA) is a strain of vaccinia virus which does not replicate in most cell types, including normal human tissues. MVA was derived by serial passage  greater than 500 times in chick embryo fibroblasts (CEF) of material derived from a pox lesion on a horse in Ankara, Turkey (Mayr et al. 1975). It was shown to be replication-impaired yet able to induce protective immunity against veterinary poxvirus infections (Mayr 1976). MVA was used as a human vaccine in the final stages of the smallpox eradication campaign, being administered by intracutaneous, subcutaneous and intramuscular routes to  greater than 120,000 subjects in southern Germany. No significant side effects were recorded, despite the deliberate targeting of vaccination to high risk groups such as those with eczema (Mayr et al. 1978; Stickl et al. 1974; Mahnel et al. 1994;). The safety of MVA reflects the avirulence of the virus in animal models, including irradiated mice and following intracranial administration to neonatal mice. The non-replication of MVA has been correlated with the production of proliferative white plaques on chick chorioallantoic membrane, abortive infection of non-avian cells, and the presence of six genomic deletions totaling approximately 30 kb (Meyer et al. 1991). The avirulence of MVA has been ascribed partially to deletions affecting host range genes K1L and C7L, although limited viral replication still occurs on human TK-143 cells and African Green Monkey CV-1 cells (Altenburger et al. 1989). Restoration of the K1L gene only partially restores MVA host range (Sutter et al. 1994). The host range restriction appears to occur during viral particle maturation, with only immature virions being observed in human HeLa cells on electron microscopy (Sutter et al. 1992). The late block in viral replication does not prevent efficient expression of recombinant genes in MVA. Recombinant MVA expressing influenza nucleoprotein, influenza haemagglutinin, and SIV proteins have proved to be immunogenic and provide varying degrees of protection in animal models, although this has never been ascribed to CD8+ T lymphocytes alone (Sutter et al. 1994, Hirsch et al. 1995; Hirsch et al. 1996). Recombinant MVA is considered a promising human vaccine candidate because of these properties of safety and immunogenicity (Moss et al. 1995). Recombinant MVA containing DNA which codes for foreign antigens is described in U.S. Pat. No. 5,185,146 (Altenburger).
Poxviruses have evolved strategies for evasion of the host immune response that include the production of secreted proteins that function as soluble receptors for tumor necrosis factor, IL-1xcex2, interferon (IFN)-xcex1/xcex2 and IFN-xcex3, which normally have sequence similarity to the extracellular domain of cellular cytokine receptors (Symons et al. 1995; Alcami et al. 1995; Alcami et al. 1992). The most recently described receptor of this nature is a chemokine receptor (Graham et al. 1997). These viral receptors generally inhibit or subvert an appropriate host immune response, and their presence is associated with increased pathogenicity. The IL-1xcex2 receptor is an exception: its presence diminishes the host febrile response and enhances host survival in the face of infection (Alcami et al. 1996). We have discovered that MVA lacks functional cytokine receptors for interferon xcex3, interferon xcex1xcex2, Tumor Necrosis Factor and CC chemokines, but it does possess the potentially beneficial IL-1xcex2 preceptor. MVA is the only known strain of vaccinia to possess this cytokine receptor profile, which theoretically renders it safer and more immunogenic than other poxviruses. Another replication-impaired and safe strain of vaccinia known as NYVAC is fully described in Tartaglia et al.(Virology 1992, 188: 217-232).
It has long been recognized that live viruses have some attractive features as recombinant vaccine vectors including a high capacity for foreign antigens and fairly good immunogenicity for cellular immune responses (Ellis 1988 new technologies for making vaccines. In: Vaccines. Editors: Plotkin S A and Mortimer E A. W B Saunders, Philadelphia, page 568; Woodrow G C 1977. In: New Generation Vaccines 2nd Edition. Editors: Levine M M, Woodrow G C, Kaper J B, Cobon G, page 33). This has led to attempts to attenuate the virulence of such live vectors in various ways including reducing their replication capacity (Tartaglia J et al. 1992 Virology 188: 217-232). However such a reduction in replication reduces the amount of antigen produced by the virus and thereby would be expected to reduce vaccine immunogenicity. Indeed attenuation of replicating vaccinia strains has previously been shown to lead to some substantial reductions in antibody responses (Lee M S et al, 1992 J Virology 66: 2617-2630). Similarly the non-replicating fowlpox vector was found to be less immunogenic for antibody production and less protective than a replicating wild-type vaccinia strain in a rabies study (Taylor J et al. 1991 Vaccine 9:190-193).
It has now been discovered that non-replicating and replication-impaired strains of poxvirus provide vectors which give an extremely good boosting effect to a primed CTL response. Remarkably, this effect is significantly stronger than a boosting effect by wild type poxviruses. The effect is observed with malarial and other antigens such as viral and tumor antigens, and is protective as shown in mice and non-human primate challenge experiments. Complete rather than partial protection from sporozoite challenge has been observed with the novel immunization regime.